Chapter 7

Cartilage and BoneCartilage and bone are both specialized connective tissues. Cartilage possesses a firm pliable matrix that resists mechanical stresses. Bone matrix is one of the hardest tissues of the body, and it too resists stresses placed upon it. Both of these connective tissues have cells that are specialized to secrete the matrix in which, subsequently, the cells become trapped. Although cartilage and bone have many varied functions, some of the functions are similar and related. Both are involved in supporting the body because they are intimately associated in the skeletal system. Most of the long bones of the body are formed first in the embryo as cartilage, which then acts as a template that is later replaced by bone; this process is referred to as endochondral bone formation. Most of the flat bones are formed within preexisting membranous sheaths; thus this method of osteogenesis is known as intramembranous bone formation.

CARTILAGE

Cartilage possesses cells called chondrocytes, which occupy small cavities called lacunae within the extracellular matrix they secreted. The substance of cartilage is neither vascularized nor supplied with nerves or lymphatic vessels; however, the cells receive their nourishment from blood vessels of surrounding connective tissues by diffusion through the matrix. The extracellular matrix is composed of glycosaminoglycans and proteoglycans, which are intimately associated with the collagen and elastic fibers embedded within the matrix. The flexibility and resistance of cartilage to compression permit it to function as a shock absorber, and its smooth surface permits almost friction-free movement of the joints of the body as it covers the articulating surfaces of the bones.

There are three types of cartilage according to the fibers present in the matrix ( Fig. 7-1 and Table 7-1 ):

Figure 7-1

Types of cartilage.

TABLE 7-1

Types of Cartilage

Type Identifying Characteristics Perichondrium Location

HyalineType II collagen, basophilic matrix, chondrocytes usually arranged in groups

Perichondrium present in most places (exceptions: articular cartilages and epiphyses)

Articular ends of long bones, nose, larynx, trachea, bronchi, ventral ends of ribs

Elastic Type II collagen, elastic fibers Perichondrium present Pinna of ear, walls of

Type Identifying Characteristics Perichondrium Locationauditory canal, auditory tube, epiglottis, cuneiform cartilage of larynx

Fibrocartilage

Type I collagen, acidophilic matrix; chondrocytes arranged in parallel rows between bundles of collagen; always associated with dense regular collagenous connective tissue or hyaline cartilage

Perichondrium absent

Intervertebral disks, articular disks, pubic symphysis, insertion of some tendons

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Hyaline cartilage contains type II collagen in its matrix; it is the most abundant cartilage in the body and serves many functions.

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Elastic cartilage contains type II collagen and abundant elastic fibers scattered throughout its matrix, giving it more pliability.

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Fibrocartilage possesses dense, coarse type I collagen fibers in its matrix, allowing it to withstand strong tensile forces.

The perichondrium is a connective tissue sheath covering that overlies most cartilage. It has an outer fibrous layer and inner cellular layer whose cells secrete cartilage matrix. The perichondrium is vascular, and its vessels supply nutrients to the cells of cartilage. In areas where the cartilage has no perichondrium (e.g., the articular surfaces of the bones forming a joint), the cartilage cells receive their nourishment from the synovial fluid that bathes the joint surfaces. Perichondria are present in elastic and most hyaline cartilages, but absent in fibrocartilage.

Hyaline Cartilage

Hyaline cartilage, the most abundant cartilage in the body, forms the template for endochondral bone formation.

Hyaline cartilage, a bluish-gray, semitranslucent, pliable substance, is the most common cartilage of the body. It is located in the nose and larynx, on the ventral ends of the ribs where they articulate with the sternum, in the tracheal rings and bronchi, and on the articulating surfaces of the movable joints of the body. Also, it is this cartilage that forms the cartilage template of many of the bones during embryonic development and constitutes the epiphyseal plates of growing bones (see Table 7-1).

Histogenesis and Growth of Hyaline Cartilage

Cells responsible for hyaline cartilage formation differentiate from mesenchymal cells.

In the region where cartilage is to form, individual mesenchymal cells retract their processes, round up, and congregate in dense masses called chondrification centers. These cells differentiate into chondroblasts and commence secreting the typical cartilage matrix around themselves. As this process continues, the chondroblasts become entrapped in their own matrix in small individual compartments called lacunae. Chondroblasts that are surrounded by this matrix are referred to as chondrocytes ( Fig. 7-2 ). These cells are still capable of cell division, forming a cluster of two to four or more cells in a lacuna. These groups are known as isogenous groups and represent one, two, or more cell divisions from an original chondrocyte (see Fig. 7-1). As the cells of an isogenous group manufacture matrix, they are pushed away from each other, forming separate lacunae and thus enlarging the cartilage from within. This type of growth is called interstitial growth.

Figure 7-2

Light micrograph of hyaline cartilage (×270). Observe the large ovoid chondrocytes (C) trapped in their lacunae. Above them are the elongated chondroblasts (Cb), and at the very top is the perichondrium (P) and the underlying chondrogenic (Cg) cell layer.

Mesenchymal cells at the periphery of the developing cartilage differentiate to form fibroblasts. These cells manufacture a dense irregular collagenous connective tissue, the perichondrium, responsible for the growth and maintenance of the cartilage. The perichondrium has two layers, an outer fibrous layer composed of type I collagen, fibroblasts, and blood vessels and an inner cellular layer composed mostly of chondrogenic cells. The chondrogenic cells undergo division and differentiate into chondroblasts, which begin to elaborate matrix. In this way cartilage also grows by adding to its periphery, a process called appositional growth.

Interstitial growth occurs only in the early phase of hyaline cartilage formation. Articular cartilage lacks a perichondrium and increases in size only by interstitial growth. This type of growth also occurs in the epiphyseal plates of long bones, where the lacunae are arranged in a longitudinal orientation parallel to the long axis of the bone; therefore, interstitial growth serves to lengthen the bone. The cartilage in the remainder of the body grows mostly by apposition, a controlled process that may continue during the life of the cartilage.

It is interesting that mesenchymal cells located within the chondrification centers are induced to become secreting chondroblasts by their attachments and the chemistry of the surrounding extracellular matrix. Also, if chondroblasts are removed from their secreted cartilage matrix and are grown in a monolayer in a low-density substrate, they will cease to secrete “cartilage matrix” containing type II collagen. Instead they will become fibroblast-like and start secreting type I collagen.

Cartilage Cells

Three types of cells are associated with cartilage: chondrogenic cells, chondroblasts, and chondrocytes (see Fig. 7-2).

Chondrogenic cells are spindle-shaped, narrow cells that are derived from mesenchymal cells. They possess an ovoid nucleus with one or two nucleoli. Their cytoplasm is sparse, and electron micrographs of chondrogenic cells display a small Golgi apparatus, a few mitochondria, some profiles of rough endoplasmic reticulum (RER), and an abundance of free ribosomes. These cells can differentiate into both chondroblasts and osteoprogenitor cells.

Chondroblasts are derived from two sources: mesenchymal cells located within the center of chondrification and chondrogenic cells of the inner cellular layer of the perichondrium (as in appositional growth). Chondroblasts are plump, basophilic cells that display the organelles required for protein synthesis. Electron micrographs of these cells demonstrate a rich network of RER, a well-developed Golgi complex, numerous mitochondria, and an abundance of secretory vesicles.

Chondrocytes are chondroblasts that are surrounded by matrix. Those near the periphery are ovoid, whereas those deeper in the cartilage are more rounded, with a diameter of 10 to 30 μm. Histological processing creates artifactual shrinkage and distortion of the cells. Chondrocytes display a large nucleus with a prominent nucleolus and the usual organelles of protein-secreting cells. Young chondrocytes have a pale-staining cytoplasm with many mitochondria, an elaborate RER, a well-developed Golgi apparatus, and glycogen. Older chondrocytes, which are relatively quiescent, display a greatly reduced complement of organelles, with an abundance of free ribosomes. Thus, these cells can resume active protein synthesis if they revert to chondroblasts.

Matrix of Hyaline Cartilage

The matrix of hyaline cartilage is composed of type II collagen, proteoglycans, glycoproteins, and extracellular fluid.

The semitranslucent blue-gray matrix of hyaline cartilage contains up to 40% of its dry weight in collagen. In addition, it contains proteoglycans, glycoproteins, and extracellular fluid. Because the refractive index of the collagen fibrils and that of the ground substance are nearly the same, the matrix appears to be an amorphous, homogeneous mass with the light microscope.

The matrix of hyaline cartilage contains primarily type II collagen, but types IX, X, and XI and other minor collagens are also present in small quantities. Type II collagen does not form large bundles, although the bundle thickness increases with distance from the lacunae. Fiber orientation appears to be related to the stresses placed on the cartilage. For example, in articular cartilage, the fibers near the surface are oriented parallel to the surface, whereas deeper fibers seem to be oriented in curved columns.

The matrix is subdivided into two regions: the territorial matrix, around each lacuna, and the interterritorial matrix (see Fig. 7-1). The territorial matrix, a 50-μm-wide band, is poor in collagen and rich in chondroitin sulfate, which contributes to its basophilic and intense staining

with periodic acid–Schiff (PAS) reagent. The bulk of the matrix is interterritorial matrix, which is richer in type II collagen and poorer in proteoglycans than the territorial matrix.

A small region of the matrix, 1- to 3-mm thick, immediately surrounding the lacuna is known as the pericellular capsule. It displays a fine meshwork of collagen fibers embedded in a basal lamina-like substance. These fibers may represent some of the other minor collagens present in hyaline cartilage; it has been suggested that the pericellular capsule may protect chondrocytes from mechanical stresses.

Cartilage matrix is rich in aggrecans, large proteoglycan molecules composed of protein cores to which glycosaminoglycan molecules (chondroitin 4-sulfate, chondroitin 6-sulfate, and heparan sulfate) are covalently linked (see Fig. 4-3). As many as 100 to 200 aggrecan molecules are linked noncovalently to hyaluronic acid, forming huge aggrecan composites that can be 3- to 4-μm long. The abundant negative charges associated with these exceedingly large proteoglycan molecules attract cations, predominantly Na + ions, which in turn attract water molecules. In this way, the cartilage matrix becomes hydrated to such an extent that up to 80% of the wet weight of cartilage is water, accounting for the ability of cartilage to resist forces of compression.

Not only do hydrated proteoglycans fill the interstices among the collagen fiber bundles, but their glycosaminoglycan side chains form electrostatic bonds with the collagen. Thus, the ground substance and fibers of the matrix form a cross-linked molecular framework that resists tensile forces.

Cartilage matrix also contains the adhesive glycoprotein chondronectin. This large molecule, similar to fibronectin, has binding sites for type II collagen, chondroitin 4-sulfate, chondroitin 6-sulfate, hyaluronic acid, and integrins (transmembrane proteins) of chondroblasts and chondrocytes. Chondronectin thus assists these cells in maintaining their contact with the fibrous and amorphous components of the matrix.

Histophysiology of Hyaline Cartilage

The smoothness of hyaline cartilage and its ability to resist forces of both compression and tension are essential to its function at the articular surfaces of joints. Because cartilage is avascular, nutrients and oxygen must diffuse through the water of hydration present in the matrix. The inefficiency of such a system necessitates a limit on the width of cartilage. There is a constant turnover in the proteoglycans of cartilage that changes with age. Hormones and vitamins also exert influence on the growth, development, and function of cartilage. Many of these substances also affect skeletal formation and growth ( Table 7-2 ).

TABLE 7-2

Effects of Hormones and Vitamins on Hyaline Cartilage

Hormone EffectThyroxine, testosterone, and somatotropin (via insulin-like growth factors)

Stimulate cartilage growth and matrix formation

Hormone Effect

Cortisone, hydrocortisone, and estradiol Inhibit cartilage growth and matrix formation

Vitamin EffectHypovitaminosis A Reduces width of epiphyseal platesHypervitaminosis A Accelerates ossification of epiphyseal plates

Hypovitaminosis C Inhibits matrix synthesis and deforms architecture of epiphyseal plate, leading to scurvy

Absence of vitamin D, resulting in deficiency in absorption of calcium and phosphorus

Proliferation of chondrocytes is normal but matrix does not become calcified properly, resulting in rickets

Hyaline cartilage degenerates when the chondrocytes hypertrophy and die and the matrix begins to calcify. This process is a normal and integral part of endochondral bone formation; however, it is also a natural process of aging, often resulting in less mobility and in joint pain.Cartilage regeneration is usually poor except in children. Chondrogenic cells from the perichondrium enter the defect and form new cartilage. If the defect is large, the cells form dense connective tissue to repair the scar.

CLINICAL CORRELATIONS

Elastic Cartilage

Elastic cartilage greatly resembles hyaline cartilage, except that its matrix and perichondrium possess elastic fibers.

Elastic cartilage is located in the pinna of the ear, the external and internal auditory tubes, the epiglottis, and the larynx (cuneiform cartilage). Because of the presence of elastic fibers, elastic cartilage is somewhat yellow and is more opaque than hyaline cartilage in the fresh state (see Table 7-1).

In most respects, elastic cartilage is identical to hyaline cartilage and is often associated with it. The outer fibrous layer of the perichondrium is rich in elastic fibers. The matrix of elastic cartilage possesses abundant, fine to coarse branching elastic fibers interposed with type II collagen fiber bundles, giving it much more flexibility than the matrix of hyaline cartilage ( Fig. 7-3 ). The chondrocytes of elastic cartilage are more abundant and larger than those of hyaline cartilage. The matrix is not as ample as in hyaline cartilage, and the elastic fiber bundles of the territorial matrix are larger and coarser than those of the interterritorial matrix.

Figure 7-3

Light micrograph of elastic cartilage (×132). Observe the perichondrium (P) and the chondrocytes (C) in their lacunae (shrunken from the walls because of processing), some of which contain more than one cell, evidence of interstitial growth. Elastic fibers (arrows) are scattered throughout.

Fibrocartilage

Fibrocartilage, unlike hyaline and elastic cartilage, does not possess a perichondrium and its matrix includes type I collagen.

Fibrocartilage is present in intervertebral disks, in the pubic symphysis, in articular disks, and attached to bone. It is associated with hyaline cartilage and with dense connective tissue, which it resembles. Unlike the other two types of cartilage, fibrocartilage does not possess a perichondrium. It displays a scant amount of matrix (rich in chondroitin sulfate and dermatan sulfate), and exhibits bundles of type I collagen, which stain acidophilic ( Fig. 7-4 ). Chondrocytes are often aligned in alternating parallel rows with the thick, coarse bundles of collagen, which parallel the tensile forces attendant on this tissue (see Table 7-1).

Figure 7-4

Light micrograph of fibrocartilage (×132). Note alignment of the chondrocytes (C) in rows interspersed with thick bundles of collagen fibers (arrows).

Chondrocytes of fibrocartilage usually arise from fibroblasts that begin to manufacture proteoglycans. As the ground substance surrounds the fibroblast, the cell becomes incarcerated in its own matrix and differentiates into a chondrocyte.

Intervertebral disks represent an example of the organization of fibrocartilage. They are interposed between the hyaline cartilage coverings of the articular surface of successive vertebrae. Each disk contains a gelatinous center, called the nucleus pulposus, which is composed of cells, derived from the notochord, lying within a hyaluronic acid-rich matrix. These cells disappear by the 20th year of life. Much of the nucleus pulposus is surrounded by the annulus fibrosus, layers of fibrocartilage whose type I collagen fibers run vertically between the hyaline cartilages of the two vertebrae. The fibers of adjacent lamellae are oriented obliquely to each other, providing support to the gelatinous nucleus pulposus. The annulus fibrosus provides resistance against tensile forces, whereas the nucleus pulposus resists forces of compression.

A ruptured disk refers to a tear or break in the laminae of the annulus fibrosus through which the gel-like nucleus pulposus extrudes. This condition occurs more often on the posterior portions of the intervertebral disks, particularly in the lumbar portion of the back, where the disk may dislocate, or slip. A “slipped disk” leads to severe, intense pain in the lower back and extremities as the displaced disk compresses the lower spinal nerves.

CLINICAL CORRELATIONS

BONE

Bone is a specialized connective tissue whose extracellular matrix is calcified, incarcerating the cells that secreted it.

Although bone is one of the hardest substances of the body, it is a dynamic tissue that constantly changes shape in relation to the stresses placed on it. For example, pressures applied to bone lead to its resorption, whereas tension applied to it results in development of new bone. In applying these facts, the orthodontist is able to remodel the bone of the dental arches by moving and straightening the teeth to correct malocclusion, thus providing the patient with a more natural and pleasant smile.

Bone is the primary structural framework for support and protection of the organs of the body, including the brain and spinal cord and the structures within the thoracic cavity, namely the lungs and heart. The bones also serve as levers for the muscles attached to them, thereby multiplying the force of the muscles to attain movement. Bone is a reservoir for several minerals of the body; for example, it stores about 99% of the body's calcium. Bone contains a central cavity, the marrow cavity, which houses the bone marrow, a hemopoietic organ.

Bone is covered on its external surface, except at synovial articulations, with periosteum, which consists of an outer layer of dense fibrous connective tissue and an inner cellular layer containing osteoprogenitor (osteogenic) cells. The central cavity of a bone is lined with endosteum, a specialized thin, connective tissue composed of a monolayer of osteoprogenitor cells and osteoblasts.

Bone is composed of cells lying in an extracellular matrix that has become calcified. The calcified matrix is composed of fibers and ground substance. The fibers constituting bone are primarily type I collagen. The ground substance is rich in proteoglycans with chondroitin sulfate and keratan sulfate side chains. In addition, glycoproteins such as osteonectin, osteocalcin, osteopontin, and bone sialoprotein are present.

The cells of bone include osteoprogenitor cells, which differentiate into osteoblasts. Osteoblasts are responsible for secreting the matrix. When these cells are surrounded by matrix, they become quiescent and are known as osteocytes. The spaces osteocytes occupy are known as lacunae ( Fig. 7-5 ). Osteoclasts, multinucleated giant cells derived from fused bone marrow precursors, are responsible for bone resorption and remodeling.

Figure 7-5

Light micrograph of decalcified compact bone (×540). Osteocytes (Oc) may be observed in lacunae (L). Also note the osteon (Os), osteoprogenitor cells (Op), and the cementing lines (Cl).

Because bone is such a hard tissue, two methods are employed to prepare it for study. Decalcified sections can be prepared by decalcifying the bone in an acid solution to remove the calcium salts. The tissue can then be embedded, sectioned, and routinely stained for study. Ground sections are prepared by sawing the bone into thin slices, followed by grinding the sections with abrasives between glass plates. When the section is sufficiently thin for study with light microscope, it is mounted for study.

Each system has disadvantages. In decalcified sections, osteocytes are distorted by the decalcifying acid bath; in ground sections, the cells are destroyed, and the lacunae and canaliculi are filled in with bone debris.

Bone Matrix

Bone matrix has inorganic and organic constituents.

Inorganic Component

The inorganic constituents of bone are crystals of calcium hydroxyapatite, composed mostly of calcium and phosphorus.

The inorganic portion of bone, which constitutes about 65% of its dry weight, is composed mainly of calcium and phosphorus along with other components, including bicarbonate, citrate, magnesium, sodium, and potassium. Calcium and phosphorus exist primarily in the form of hydroxyapatite crystals [Ca 10(PO 4) 6(OH) 2], but calcium phosphate is also present in an amorphous form. Hydroxyapatite crystals (40-nm long by 25-nm wide by 1.5- to 3-nm thick) are arranged in an ordered fashion along the type I collagen fibers; they are deposited into the gap regions of the collagen but also are present along the overlap region. The free surface of the crystals is surrounded by amorphous ground substance. The surface ions of the crystals attract H 2O and form a hydration shell, which permits ion exchange with the extracellular fluid.

Bone is one of the hardest and strongest substances in the body. Its hardness and strength are due to the association of hydroxyapatite crystals with collagen. If bone is decalcified (i.e., all of the mineral is removed from the bone), it still retains its original shape but becomes so flexible that it can be bent like a piece of tough rubber. If the organic component is extracted from bone, the mineralized skeleton still retains its original shape, but it becomes extremely brittle and can be fractured with ease.

Organic Component

The predominant organic component of bone is type I collagen.

The organic component of bone matrix, constituting approximately 35% of the dry weight of bone, includes fibers that are almost exclusively type I collagen.

Collagen, most of which is type I, makes up about 80% to 90% of the organic component of bone. It is formed in large (50 to 70 nm in diameter) bundles displaying a typical 67-nm periodicity. Type I collagen in bone is highly cross-linked, which prevents it from being easily extracted.

The fact that bone matrix stains with PAS reagent and displays slight metachromasia indicates the presence of sulfated glycosaminoglycans, namely chondroitin sulfate and keratan sulfate. These form small proteoglycan molecules with short protein cores to which the glycosaminoglycans are covalently bound. The proteoglycans are noncovalently bound, via link

proteins, to hyaluronic acid, forming very large aggrecan composites. The abundance of collagen, however, causes the matrix to be acidophilic.

Several glycoproteins are also present in the bone matrix. These appear to be restricted to bone and include osteocalcin, which binds to hydroxyapatite, and osteopontin, which also binds to hydroxyapatite but has additional binding sites for other components as well as for integrins present on osteoblasts and osteoclasts. Vitamin D stimulates the synthesis of these glycoproteins. Bone sialoprotein, another matrix protein, has binding sites for matrix components and integrins of osteoblasts and osteocytes, suggesting its involvement in the adherence of these cells to bone matrix.

Cells of Bone

The cells of bone are osteoprogenitor cells, osteoblasts, osteocytes, and osteoclasts.

Osteoprogenitor Cells

Osteoprogenitor cells are derived from embryonic mesenchymal cells and retain their ability to undergo mitosis.

Osteoprogenitor cells are located in the inner cellular layer of the periosteum, lining haversian canals, and in the endosteum (see Fig. 7-5). These cells, derived from embryonic mesenchyme, remain in place throughout postnatal life and can undergo mitotic division and have the potential to differentiate into osteoblasts. Moreover, under certain conditions of low oxygen tension, these cells may differentiate into chondrogenic cells. Osteoprogenitor cells are spindle-shaped and have a pale-staining oval nucleus; their scant pale-staining cytoplasm displays sparse RER and a poorly developed Golgi apparatus but an abundance of free ribosomes. These cells are most active during the period of intense bone growth.

Osteoblasts

Osteoblasts not only synthesize the organic matrix of bone but also possess receptors for parathyroid hormone.

Osteoblasts are derived from osteoprogenitor cells and develop under the influence of the bone morphogenic protein (BMP) family and transforming growth factor-β. Osteoblasts are responsible for the synthesis of the organic protein components of the bone matrix, including type I collagen, proteoglycans, and glycoproteins. Additionally, they produce RANKL (receptor for activation of nuclear factor kappa B), osteocalcin (for bone mineralization), osteopontin (for formation of sealing zone between osteoclasts and the subosteoclastic compartment), osteonectin (related to bone mineralization), bone sialoprotein (binding osteoblasts to extracellular matrix), and macrophage colony-stimulating factor (M-CSF) (discussed later). Osteoblasts are located on the surface of the bone in a sheet-like arrangement of cuboidal to columnar cells ( Fig. 7-6 ). When actively secreting matrix, they exhibit a basophilic cytoplasm.

Figure 7-6

Light micrograph of intramembranous ossification (×540). Osteoblasts (Ob) line the bony spicule where they are secreting osteoid onto the bone. Osteoclasts (Oc) may be observed housed in Howship's lacunae.

The organelles of osteoblasts are polarized so that the nucleus is located away from the region of secretory activity, which houses secretory granules believed to contain matrix precursors. The contents of these vesicles stain pink with PAS reagent.

Electron micrographs exhibit abundant RER, a well-developed Golgi complex ( Fig. 7-7A ), and numerous secretory vesicles containing flocculent material that accounts for the PAS pink-staining vacuoles observed in the light microscope. Osteoblasts extend short processes that make contact with those of neighboring osteoblasts, as well as long processes that make contact with processes of osteocytes. Although these processes form gap junctions with one another, the number of gap junctions between osteoblasts is much fewer than those between osteocytes.

Figure 7-7

Electron micrographs of bone-forming cells. A, Five osteoblasts (1 to 5) lined up on the surface of bone (B) displaying abundant rough endoplasmic reticulum. Observe the process of an osteocyte in a canaliculus ( arrow). The cell with the elongated nucleus lying above the osteoblasts is an osteoprogenitor cell (Op) (×2500). B, Note the osteocyte in its lacuna (L) with its processes extending into canaliculi (arrows) (×1000). B, bone; C, cartilage.

(From Marks SC Jr, Popoff SN: Bone cell biology: The regulation of development, structure, and function in the skeleton. Am J Anat 183:1-44, 1988.)

Osteoblast cell membranes are rich in the enzyme alkaline phosphatase. During active bone formation, these cells secrete high levels of alkaline phosphatase, elevating the levels of this enzyme in the blood. Thus, the clinician can monitor bone formation by measuring the blood alkaline phosphatase level.

CLINICAL CORRELATIONS

As osteoblasts exocytose their secretory products, each cell surrounds itself with the bone matrix it has just produced; when this occurs, the incarcerated cell is referred to as an osteocyte, and the space it occupies is known as a lacuna. Most of the bone matrix becomes calcified; however, osteoblasts as well as osteocytes are always separated from the calcified substance by a thin, noncalcified layer known as the osteoid (uncalcified bone matrix).

Surface osteoblasts that cease to form matrix revert to a more flattened-shaped quiescent state and are called bone-lining cells. Although these cells appear to be similar to osteoprogenitor cells, they are most likely incapable of dividing but can be reactivated to the secreting form with the proper stimulus.

Osteoblasts have several factors on their cell membranes, the most significant of which are integrins and parathyroid hormone receptors. When parathyroid hormone binds to these

receptors, it stimulates osteoblasts to secrete osteoprotegerin ligand (OPGL), a factor that induces the differentiation of preosteoclasts into osteoclasts and it increases RANKL expression. Also osteoblasts secrete an osteoclast-stimulating factor, which activates osteoclasts to resorb bone. Osteoblasts also secrete enzymes responsible for removing osteoid so that osteoclasts can make contact with the mineralized bone surface.

Osteocytes

Osteocytes are mature bone cells derived from osteoblasts that became trapped in their lacunae.

Osteocytes are mature bone cells, derived from osteoblasts, that are housed in lacunae within the calcified bony matrix (see Figure 7-5 Figure 7-7 ). There are as many as 20,000 to 30,000 osteocytes per mm 3 of bone. Radiating out in all directions from the lacunaa are narrow, tunnel-like spaces (canaliculi) that house cytoplasmic processes of the osteocyte. These processes make contact with similar processes of neighboring osteocytes, forming gap junctions through which ions and small molecules can move between the cells. The canaliculi also contain extracellular fluid carrying nutrients and metabolites that nourish the osteocytes.

Osteocytes conform to the shape of their lacunae. Their nucleus is flattened, and their cytoplasm is poor in organelles, displaying scant RER and a greatly reduced Golgi apparatus. Although osteocytes appear to be inactive cells, they secrete substances necessary for bone maintenance. These cells have also been implicated in mechanotransduction, in that they respond to stimuli that place tension on bone by releasing cyclic adenosine monophosphate (cAMP), osteocalcin, and insulin-like growth factor. The release of these factors facilitates the recruitment of preosteoblasts to assist in the remodeling of the skeleton (adding more bone) not only during growth and development but also during the long-term redistribution of forces acting on the skeleton. An example of such remodeling is evident in the comparison of male and female skeletons, in which the muscle attachments of the male skeleton are usually better defined than those of the female skeleton.

The interval between the osteocyte plasmalemma and the walls of the lacunae and canaliculi, known as the periosteocytic space, is occupied by extracellular fluid. Considering the extensive network of the canaliculi and the sheer number of osteocytes present in the skeleton of an average person, the volume of the periosteocytic space and the surface area of the walls have been calculated to be a staggering 1.3 L and as much as 5000 m 2, respectively. It has been suggested that the 1.3 L of extracellular fluid occupying the periosteocytic space is exposed to as much as 20 g of exchangeable calcium that can be resorbed from the walls of these spaces. The resorbed calcium gains access to the bloodstream and ensures the maintenance of adequate blood calcium levels.

Osteoclasts

Osteoclasts are multinucleated cells originating from granulocyte-macrophage progenitors. They play a role in bone resorption.

The precursor of the osteoclast originates in the bone marrow. Osteoclasts have receptors for osteoclast-stimulating factor, colony-stimulating factor-1, osteoprotegerin (OPG), and calcitonin, among others. Osteoclasts are responsible for resorbing bone, and after they finish doing so, these cells probably undergo apoptosis.

Morphology of Osteoclasts

Osteoclasts are large, motile, multinucleated cells 150 μm in diameter; they contain up to 50 nuclei and have an acidophilic cytoplasm (see Fig. 7-6). Osteoclasts were once thought to be derived from the fusion of many blood-derived monocytes, but the newest evidence shows that they have a bone marrow precursor in common with monocytes termed the mononuclear-phagocyte system. These precursor cells are stimulated by macrophage colony–stimulating factor to undergo mitosis. In the presence of bone, these osteoclast precursors fuse to produce the multinucleated osteoclasts.

Osteoblasts secrete three signaling molecules that regulate the differentiation of osteoclasts. The first of these signals, the macrophage colony–stimulating factor (M-CSF) binds to a receptor on the macrophage, inducing it to become a proliferating osteoclast precursor, and it induces the expression of the receptor for activation of nuclear factor kappa B (RANK) on the precursor. Another osteoblast signaling molecule, RANKL, binds to the RANKL receptor on the osteoclastic precursor, inducing it to differentiate into the multinucleated osteoclast, activating it, and enhancing bone resorption. The third signaling molecule, OPG, a member of the tumor necrosis factor receptor (TNFR) family, can serve as a decoy by interacting with RANKL, thus prohibiting it from binding to the macrophage and thus inhibiting osteoclast formation. In this way, RANKL, RANK, and OPG regulate bone metabolism and osteoclastic activity. OPG is produced not only by osteoblasts but by cells of many other tissues, including the cardiovascular system, lung, kidney, intestines, as well as hematopoietic and immune cells. Therefore it is not surprising that its expression is modulated by various means by cytokines, peptides, hormones, drugs, and so forth. In bone, OPG not only inhibits the differentiation of precursor cells into osteoclasts but also suppresses the osteoclast's bone resorptive capacities. Also, tensional forces on bone trigger OPG and mRNA synthesis.

Osteoclasts occupy shallow depressions, called Howship's lacunae, that identify regions of bone resorption. An osteoclast active in bone resorption may be subdivided into four morphologically recognizable regions:

1

The basal zone, located farthest from the Howship lacunae, houses most of the organelles, including the multiple nuclei and their associated Golgi complexes and centrioles. Mitochondria, RER, and polysomes are distributed throughout the cell but are more numerous near the ruffled border.

2

The ruffled border is the portion of the cell that is directly involved in resorption of bone. Its finger-like processes are active and dynamic, changing their configuration continuously as they project into the resorption compartment, known as the subosteoclastic compartment. The cytoplasmic aspect of the ruffled border plasmalemma displays a regularly spaced, bristle-like coat that increases the thickness of the plasma membrane of this region.

3

The clear zone is the region of the cell that immediately surrounds the periphery of the ruffled border. It is organelle-free but contains many actin microfilaments that form an actin ring and appear to function in helping integrins of the clear zone plasmalemma maintain contact with the bony periphery of the Howship lacunae. In fact, the plasma membrane of this region is so closely applied to the bone that it forms the sealing zone of the subosteoclastic compartment. Thus, the clear zone isolates the subosteoclastic compartment from the surrounding region, establishing a microenvironment whose contents may be modulated by cellular activities. For the osteoclast to be able to resorb bone, the actin ring must first be formed, and its formation may be facilitated by OPGL. Then the ruffled border is formed, whose finger-like processes increase the surface area of the plasmalemma in the region of bone resorption, facilitating the resorptive process.

4

The vesicular zone of the osteoclast consists of numerous endocytotic and exocytotic vesicles that ferry lysosomal enzymes and metalloproteinases into the subosteoclastic compartment and the products of bone degradation into the cell ( Fig. 7-8 ). The vesicular zone is between the basal zone and the ruffled border.

Figure 7-8

Electron micrograph of an osteoclast. Note the clear zone (Cz) on either side of the ruffled border (B) of this multinucleated cell.

(From Marks SC Jr, Walker DG: The hematogenous origin of osteoclasts. Experimental evidence from osteopetrotic [microphthalmic] mice treated with spleen cells from beige mouse donors. Am J Anat 161:1-10, 1981.)

Mechanism of Bone Resorption

Within osteoclasts, the enzyme carbonic anhydrase catalyzes the intracellular formation of carbonic acid (H 2CO 3) from carbon dioxide and water. Carbonic acid dissociates within the cells into H + ions and bicarbonate ions, HCO 3 −. The bicarbonate ions, accompanied by Na + ions, cross the plasmalemma and enter nearby capillaries. Proton pumps in the plasmalemma of the ruffled border of the osteoclasts actively transport H + ions into the subosteoclastic compartment, reducing the pH of the microenvironment (Cl − ions follow passively). The

inorganic component of the matrix is dissolved as the environment becomes acidic; the liberated minerals enter the osteoclast cytoplasm to be delivered to nearby capillaries.

Lysosomal hydrolases and metalloproteinases, such as collagenase and gelatinase, are secreted by osteoclasts into the subosteoclastic compartment to degrade the organic components of the decalcified bone matrix. The degradation products are endocytosed by the osteoclasts and further broken down into amino acids, monosaccharides, and disaccharides, which then are released into nearby capillaries ( Fig. 7-9 ).

Figure 7-9

Osteoclastic function. RER, rough endoplasmic reticulum.

(From Gartner LP, Hiatt JL, Strum JM: Cell Biology and Histology [Board Review Series]. Philadelphia, Lippincott Williams & Wilkins, 1998, p 100.)

Osteopetrosis, not to be confused with osteoporosis, is a genetic disorder where osteoclasts do not possess a ruffled border. Consequently, these osteoclasts cannot resorb bone and persons with osteopetrosis display increased bone density. Individuals suffering from this disease may exhibit anemia resulting from decreased marrow space, as well as blindness, deafness, and cranial nerve involvement because of impingement of the nerves due to narrowing of the foramina.

CLINICAL CORRELATIONS

Hormonal Control of Bone Resorption

The bone-resorbing activity of osteoclasts is regulated by two hormones, parathyroid hormone and calcitonin, produced by the parathyroid and thyroid gland, respectively.

Bone Structure

Bones are classified according to their anatomical shape: long, short, flat, irregular, and sesamoid.

Bones are classified according to their shape:

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Long bones display a shaft located between two heads (e.g., tibia).

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Short bones have more or less the same width and length (e.g., carpal bones of the wrist).

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Flat bones are flat, thin, and plate-like (e.g., bones forming the brain case of the skull).

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Irregular bones have an irregular shape that does not fit into the other classes (e.g., sphenoid and ethmoid bones within the skull).

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Sesamoid bones develop within tendons, where they increase the mechanical advantage for the muscle (e.g., patella) across a joint.

Gross Observation of Bone

Gross observations of the femur (a long bone) cut in longitudinal section reveal two different types of bone structure. The very dense bone on the outside surface is compact bone, whereas the porous portion lining the marrow cavity is cancellous or spongy bone ( Fig. 7-10 ). Closer observation of the spongy bone reveals branching bony trabeculae and spicules jutting out from the internal surface of the compact bone into the marrow cavity. There are no haversian systems in spongy bone, but there are irregular arrangements of lamellae. These contain lacunae housing osteocytes that are nourished by diffusion from the marrow cavity, which is filled with bone marrow.

Figure 7-10

Diagram of bone illustrating compact cortical bone, osteons, lamellae, Volkmann's canals, haversian canals, lacunae, canaliculi, and spongy bone.

Bone marrow exists as two types: red bone marrow, in which blood cells are forming, and yellow bone marrow, composed mostly of fat.

The shaft of a long bone is called the diaphysis, and the articular ends are called the epiphyses (singular, epiphysis). In a person who is still growing, the diaphysis is separated from each epiphysis by the epiphyseal plate of cartilage. The articular end of the bone is enlarged and sculpted to articulate with its bony counterpart of the joint. The surface of the articulating end is covered with only a thin layer of compact bone overlying spongy bone. On top of this is the highly polished articular hyaline cartilage, which reduces friction as it moves against the articular cartilage of the bony counterpart of the joint. The area of transition between the epiphyseal plate and the diaphysis is called the metaphysis, where columns of spongy bone are located. It is from the epiphyseal plate and the metaphysis that bone grows in length.

The diaphysis is covered by a periosteum except where tendons and muscles insert into the bone. There is no periosteum on the surfaces of bone covered by articular cartilage. Periosteum is also absent from sesamoid bones (e.g., patella), which are formed within tendons and function

to increase the mechanical advantage across a joint. The periosteum is a noncalcified, dense, irregular, collagenous connective tissue covering the bone on its external surface and inserting into it via Sharpey's fibers (see Fig. 7-10). Periosteum is composed of two layers. The outer fibrous layer helps distribute vascular and nerve supply to bone, whereas the inner cellular layer possesses osteoprogenitor cells and osteoblasts.

The flat bones of the skull develop by a method different from that of most of the long bones of the body. The inner and outer surfaces of the calvaria (skull cap) possess two relatively thick layers of compact bone called the inner and outer tables, which surround the spongy bone (diploë) sandwiched between them. The outer table possesses a periosteum, identified as the pericranium, whereas internally the inner table is lined with dura mater, which serves as a periosteum for the inner table and as a protective covering for the brain.

Bone Types Based on Microscopic Observations

Microscopically, bone is classified as either primary (immature) or secondary (mature) bone.

Microscopic observations reveal two types of bone: primary bone, or immature or woven bone, and secondary bones, or mature or lamellar bone.

Primary bone is immature in that it is the first bone to form during fetal development and during bone repair. It has abundant osteocytes and irregular bundles of collagen, which are later replaced and organized as secondary bone except in certain areas (e.g., at sutures of the calvaria, insertion sites of tendons, and bony alveoli surrounding the teeth). The mineral content of primary bone is also much less than that of secondary bone.

Secondary bone is mature bone composed of parallel or concentric bony lamellae 3- to 7-μm thick. Osteocytes in their lacunae are dispersed at regular intervals between, or occasionally within, lamellae. Canaliculi, housing osteocytic processes, connect neighboring lacunae with one another, forming a network of intercommunicating channels that facilitate the flow of nutrients, hormones, ions, and waste products to and from osteocytes. In addition, osteocytic processes within these canaliculi make contact with similar processes of neighboring osteocytes and form gap junctions, permitting these cells to communicate with each other.

Because the matrix of secondary bone is more calcified, it is stronger than primary bone. In addition, the collagen fibers of secondary bone are arranged so that they parallel each other within a given lamella.

Lamellar Systems of Compact Bone

There are four lamellar systems in compact bone: outer circumferential lamellae, inner circumferential lamellae, osteons, and interstitial lamellae.

Compact bone is composed of wafer-like thin layers of bone, lamellae, that are arranged in lamellar sys-tems that are especially evident in the diaphyses of long bones. These lamellar

systems are the outer circumferential lamellae, inner circumferential lamellae, osteons (haversian canal systems), and interstitial lamellae.

OUTER AND INNER CIRCUMFERENTIAL LAMELLAE

The outer circumferential lamellae are just deep to the periosteum, forming the outermost region of the diaphysis, and contain Sharpey's fibers anchoring the periosteum to the bone (see Fig. 7-10).

The inner circumferential lamellae, analogous to but not as extensive as outer circumferential lamellae, completely encircle the marrow cavity. Trabeculae of spongy bone extend from the inner circumferential lamellae into the marrow cavity, interrupting the endosteal lining of the inner circumferential lamellae.

HAVERSIAN CANAL SYSTEMS (OSTEONS)

The bulk of compact bone is composed of an abundance of haversian canal systems (osteons); each system is composed of cylinders of lamellae, concentrically arranged around a vascular space known as the haversian canal ( Fig. 7-11 ; also see Fig. 7-10). Frequently, osteons bifurcate along their considerable length. Each osteon is bounded by a thin cementing line, composed mostly of calcified ground substance with a scant amount of collagen fibers (see Fig. 7-5).

Figure 7-11

Light micrograph of undecalcified ground bone (×270). Observe the haversian system containing the haversian canal (C) and concentric lamellae (L) with lacunae with their canaliculi (arrows).

Collagen fiber bundles are parallel to each other within a lamella but are oriented almost perpendicular to those of adjacent lamellae. This arrangement is possible because the collagen fibers follow a helical arrangement around the haversian canal within each lamella but are pitched differently in adjacent lamellae.

Each haversian canal, lined by a layer of osteoblasts and osteoprogenitor cells, houses a neurovascular bundle with its associated connective tissue. Haversian canals of adjacent osteons are connected to each other by Volkmann's canals ( Fig. 7-12 ; also see Fig. 7-10). These vascular spaces are oriented oblique to or perpendicular to haversian canals.

Figure 7-12

Light micrograph of decalcified compact bone (×162). Several osteons (Os) are displayed with their concentric lamellae (L). A Volkmann's canal (V) is also displayed. The dark-staining structures scattered throughout represent nuclei of osteocytes (Oc).

The diameter of haversian canals varies from approximately 20 μm to about 100 μm. During the formation of osteons, the lamella closest to the cementing line is the first one to be formed. As additional lamellae are added to the system, the diameter of the haversian canal is reduced, and

the thickness of the osteon wall increases. Because nutrients from blood vessels of the haversian canal must traverse canaliculi to reach osteocytes, an inefficient process, most osteons possess only 4 to 20 lamellae.

As bone is being remodeled, osteoclasts resorb osteons and osteoblasts replace them. Remnants of osteons remain as irregular arcs of lamellar fragments, known as interstitial lamellae, surrounded by osteons. Like osteons, interstitial lamellae are also surrounded by cementing lines.

Histogenesis of Bone

Bone formation during embryonic development may be of two types: intramembranous and endochondral. Bone that is formed by either of the two methods is identical histologically. The first bone formed is primary bone, which is later resorbed and replaced by secondary bone. Secondary bone continues to be resorbed throughout life, although at a slower rate.

Intramembranous Bone Formation

Intramembranous bone formation occurs within mesenchymal tissue.

Most flat bones are formed by intramembranous bone formation. This process occurs in a richly vascularized mesenchymal tissue, whose cells make contact with each other via long processes.

Mesenchymal cells differentiate into osteoblasts that secrete bone matrix, forming a network of spicules and trabeculae whose surfaces are populated by these cells ( Figure 7-13 Figure 7-14 ). This region of initial osteogenesis is known as the primary ossification center. The collagen fibers of these developing spicules and trabeculae are randomly oriented, as expected in primary bone. Calcification quickly follows osteoid formation, and osteoblasts trapped in their matrices become osteocytes. The processes of these osteocytes are also surrounded by forming bone, establishing a system of canaliculi. Continuous mitotic activity of mesenchymal cells provides a supply of undifferentiated osteoprogenitor cells, which form osteoblasts.

Figure 7-13

Intramembranous bone formation.

Figure 7-14

Light micrograph of intramembranous bone formation (ossification) (×132). Trabeculae of bone are being formed by osteoblasts lining their surface (arrows) Observe osteocytes trapped in lacunae (arrowheads). Primitive osteons (Os) are beginning to form.

As the sponge-like network of trabeculae is established, the vascular connective tissue in their interstices is transformed into bone marrow. The addition of trabeculae to the periphery increases the size of the forming bone. Larger bones, such as the occipital bone of the base of the skull, have several ossification centers, which fuse with each other to form a single bone. The

fontanelles (“soft spots”) on the frontal and parietal bones of a newborn infant represent ossification centers that are not fused prenatally.

Regions of the mesenchymal tissues that remain uncalcified differentiate into the periosteum and endosteum of developing bone. Moreover, the spongy bone deep to the periosteum and the periosteal layer of the dura mater of flat bones are transformed into compact bone, forming the inner and outer tables with the intervening diploë.

Endochondral Bone Formation

Endochondral bone formation requires the presence of a cartilage template.

Most of the long and short bones of the body develop by endochondral bone formation ( Table 7-3 ). This type of bone formation occurs in several phases, the most critical of which are (1) formation of a miniature hyaline cartilage model, (2) continued growth of the model, which serves as a structural scaffold for bone development, and (3) eventual resorption and replacement by bone ( Fig. 7-15 ).

TABLE 7-3

Events in Endochondral Bone Formation

Event Description

Hyaline cartilage model formed.

Miniature hyaline cartilage model formed in region of developing embryo where bone is to develop; some chondrocytes mature, hypertrophy, and die; cartilage matrix becomes calcified

Primary Center of OssificationPerichondrium at the midriff of diaphysis becomes vascularized.

Vascularization of perichondrium changes it to periosteum Chondrogenic cells become osteoprogenitor cells

Osteoblasts secrete matrix, forming subperiosteal bone collar.

The subperiosteal bone collar is formed of primary bone (intramembranous bone formation)

Chondrocytes within the diaphysis core hypertrophy die, and degenerate.

Presence of periosteum and bone prevents diffusion of nutrients to chondrocytes; their degeneration leaves lacunae, opening large spaces in septa of cartilage

Osteoclasts etch holes in subperiosteal bone collar, permitting entrance of osteogenic bud

Holes permit osteoprogenitor cells and capillaries to invade cartilage model, now calcified, and begin elaborating bone matrix

Calcified cartilage/calcified bone complex is formed.

Bone matrix laid down on septa of calcified cartilage forms this complex (histologically, calcified cartilage stains blue, calcified bone stains red)

Osteoclasts begin resorbing the calcified cartilage/calcified bone complex.

Destruction of the calcified cartilage/calcified bone complex enlarges the marrow cavity

Subperiosteal bone collar thickens, This event, over a period of time, completely replaces

Event Descriptionbegins growing toward the epiphyses. diaphyseal cartilage with bone

Secondary Center of Ossification

Ossification begins at epiphysis.Process begins in same way as at primary center, except that there is no bone collar; osteoblasts lay down bone matrix on calcified cartilage scaffold

Growth of bone occurs at epiphyseal plate.

Cartilaginous articular surface of bone remains; epiphyseal plate persists—growth added at epiphyseal end of plate. Bone is added at diaphyseal end of plate

Epiphysis and diaphysis become continuous.

At the end of bone growth, cartilage of epiphyseal plate ceases proliferation; bone development continues to unite the diaphysis and epiphysis

Figure 7-15

Endochondral bone formation. Blue represents the cartilage model upon which bone is formed. The bone then replaces the cartilage. A, Hyaline cartilage model. B, Cartilage at the midriff (diaphysis) is invaded by vascular elements. C, Subperiosteal bone collar is formed. D, Bone collar prevents nutrients from reaching cartilage cells so they die leaving confluent lacunae. Osteoclasts invade and etch bone to permit periosteal bud to form. E, Calcified bone/calcified cartilage complex at epiphyseal ends of the growing bone. F, Enlargement of the epiphyseal plate at the end of the bone where bone replaces cartilage.

EVENTS OCCURRING AT THE PRIMARY CENTER OF OSSIFICATION

1

In the region where bone is to grow within the embryo, a hyaline cartilage model of that bone develops. This event begins in exactly the same way that hyaline cartilage at any location would develop (discussed earlier). For a period this model grows both appositionally and interstitially. Eventually, the chondrocytes in the center of the cartilage model hypertrophy, accumulate glycogen in their cytoplasm, and become vacuolated ( Fig. 7-16 ). Hypertrophy of the chondrocytes results in enlargement of their lacunae and reduction in the intervening cartilage matrix septae, which become calcified.

Figure 7-16

Electron micrograph of hypertrophic chondrocytes in the growing mandibular condyle (×83,000). Observe the abundant rough endoplasmic reticulum and developing Golgi apparatus (G). Note also glycogen (gly) deposits in one end of the cells, a characteristic of these cells shortly before death. Col, collagen fibers; Fw, territorial matrix.

(From Marchi F, Luder HU, Leblond CP: Changes in cells' secretory organelles and extracellular matrix during endochondral ossification in the mandibular condyle of the growing rat. Am J Anat 190:41-73, 1991.)

2

Concurrently, the perichondrium at the midriff of the diaphysis of cartilage becomes vascularized ( Fig. 7-17 ). When this happens, chondrogenic cells become osteoprogenitor cells forming osteoblasts, and the overlying perichondrium becomes a periosteum.

Figure 7-17

Light micrograph of endochondral bone formation (×14). The upper half demonstrates cartilage (C) containing chondrocytes that mature, hypertrophy, and calcify at the interface; the lower half shows where calcified cartilage/bone complex (arrows) is being resorbed and bone (b) is being formed. P, periosteum.

3

The newly formed osteoblasts secrete bone matrix, forming the subperiosteal bone collar on the surface of the cartilage template by intramembranous bone formation (see Fig. 7-17).

4

The bone collar prevents the diffusion of nutrients to the hypertrophied chondrocytes within the core of the cartilage model, causing them to die. This process is responsible for the presence of empty, confluent lacunae forming large concavities—the future marrow cavity in the center of the cartilage model.

5

Holes etched in the bone collar by osteoclasts permit a periosteal bud (osteogenic bud), composed of osteoprogenitor cells, hemopoietic cells, and blood vessels, to enter the concavities within the cartilage model (see Fig. 7-15).

6

Osteoprogenitor cells divide to form osteoblasts. These newly formed cells elaborate bone matrix on the surface of the calcified cartilage. The bone matrix becomes calcified to form a calcified cartilage/calcified bone complex. This complex can be appreciated in routinely stained histological sections because calcified cartilage stains basophilic, whereas calcified bone stains acidophilic ( Fig. 7-18 Fig. 7-19 ).

Figure 7-18

Light micrograph of endochondral bone formation (×132). Observe the blood vessel (BV), bone-covered trabeculae (Tr) of calcified cartilage, and medullary cavity (MC).

Figure 7-19

Higher magnification of endochondral bone formation (×270). The trabeculae of calcified cartilage (CC) are covered by a thin layer of bone ( darker red) with osteocytes embedded in it ( arrows) and with osteoblasts (Ob) lying next to the bone.

7

As the subperiosteal bone becomes thicker and grows in each direction from the midriff of the diaphysis toward the epiphyses, osteoclasts begin resorbing the calcified cartilage/calcified bone complex, enlarging the marrow cavity. As this process continues, the cartilage of the diaphysis is replaced by bone except for the epiphyseal plates, which are responsible for the continued growth of the bone for 18 to 20 years.

EVENTS OCCURRING AT SECONDARY CENTERS OF OSSIFICATION

Secondary centers of ossification begin to form at the epiphysis at each end of the forming bone by a process similar to that in the diaphysis, except that a bone collar is not formed. Rather, osteoprogenitor cells invade the cartilage of the epiphysis, differentiate into osteoblasts, and begin secreting matrix on the cartilage scaffold (see Fig. 7-15). These events take place and progress much as they do in the diaphysis, and eventually the cartilage of the epiphysis is replaced with bone except at the articular surface and at the epiphyseal plate. The articular surface of the bone remains cartilaginous throughout life. The process at the epiphyseal plate, which controls bone length, is described in the next section.

These events are a dynamic continuum that is completed over a number of years as bone growth and development progress toward the growing epiphyses at each end of the bone (see Table 7-3). At the same time, the bone is constantly being remodeled to meet the changing forces placed on it.

BONE GROWTH IN LENGTH

The continued lengthening of bone depends on the epiphyseal plate.

The chondrocytes of the epiphyseal plate proliferate and participate in the process of endochondral bone formation. Proliferation occurs at the epiphyseal aspect, and replacement by bone takes place at the diaphyseal side of the plate. Histologically, the epiphyseal plate is divided into five recognizable zones. These zones, beginning at the epiphyseal side, are as follows:

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Zone of reserve cartilage: Chondrocytes randomly distributed throughout the matrix are mitotically active.

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Zone of proliferation: Chondrocytes, rapidly proliferating, form rows of isogenous cells that parallel the direction of bone growth.

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Zone of maturation and hypertrophy: Chondrocytes mature, hypertrophy, and accumulate glycogen in their cytoplasm (see Fig. 7-16). The matrix between their lacunae narrows with a corresponding growth of lacunae.

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Zone of calcification: Lacunae become confluent, hypertrophied chondrocytes die, and cartilage matrix becomes calcified.

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Zone of ossification: Osteoprogenitor cells invade the area and differentiate into osteoblasts, which elaborate matrix that becomes calcified on the surface of calcified cartilage. This is followed by resorption of the calcified cartilage/calcified bone complex.

As long as the rate of mitotic activity in the zone of proliferation equals the rate of resorption in the zone of ossification, the epiphyseal plate remains the same width and the bone continues to grow longer. At about the 20th year of age, the rate of mitosis decreases in the zone of proliferation and the zone of ossification overtakes the zones of proliferation and cartilage reserve. The cartilage of the epiphyseal plate is replaced by a plate of calcified cartilage/calcified bone complex, which is resorbed by osteoclastic activity, and the marrow cavity of the diaphysis becomes confluent with the bone marrow cavity of the epiphysis. Once the epiphyseal plate is resorbed, growth in length is no longer possible.

BONE GROWTH IN WIDTH

Bone growth in width takes place by appositional growth.

The events just described detail how bone lengthening is accomplished by the proliferation and interstitial growth of cartilage, which is eventually replaced by bone. Growth of the diaphysis in girth, however, takes place by appositional growth. The osteoprogenitor cells of the osteogenic layer of the periosteum proliferate and differentiate into osteoblasts that begin elaborating bone matrix on the subperiosteal bone surface. This process occurs continuously throughout the total period of bone growth and development, so that in a mature long bone the shaft is built via subperiosteal intramembranous bone formation.

During bone growth and development, bone resorption is as important as bone deposition. Formation of bone on the outside of the shaft must be accompanied by osteoclastic activity internally so that the marrow space can be enlarged.

Calcification of Bone

Calcification begins when there are deposits of calcium phosphate on the collagen fibril.

Exactly how calcification occurs is unclear, although it is known to be stimulated by certain proteoglycans and the Ca 2+-binding glycoprotein osteonectin as well as bone sialoprotein. One theory, called heterogeneous nucleation, is that collagen fibers in the matrix are nucleation sites for the metastable calcium and phosphate solution and that the solution begins to crystallize into the gap region of the colla-gen. Once this region has “nucleated,” calcification proceeds.

The most commonly accepted theory of calcification is based on the presence of matrix vesicles within the osteoid. Osteoblasts release these small, membrane-bounded matrix vesicles, 100 to 200 nm in diameter, which contain a high concentration of Ca 2+ and PO 4 3 ions, cAMP, adenosine triphosphate (ATP), adenosine triphosphatase (ATPase), alkaline phosphatase, pyrophosphatase, calcium-binding proteins, and phosphoserine. The matrix vesicle membrane possesses numerous calcium pumps, which transport Ca 2− ions into the vesicle. As the concentration of calcium Ca 2− ions within the vesicle increases, crystallization occurs and the growing calcium hydroxyapatite crystal pierces the membrane, bursting the matrix vesicle and releasing its contents.

Alkaline phosphatase cleaves pyrophosphate groups from the macromolecules of the matrix. The liberated pyrophosphate molecules are inhibitors of calcification, but they are cleaved by the enzyme pyrophosphatase into PO 4 3− ions, increasing the concentration of this ion in the microenvironment.

The calcium hydroxyapatite crystals released from the matrix vesicles act as nidi of crystallization. The high concentration of ions in their vicinity, along with the presence of calcification factors and calcium-binding proteins, fosters the calcification of the matrix. As crystals are deposited into the gap regions on the surface of collagen molecules, water is resorbed from the matrix.

Mineralization occurs around numerous closely spaced nidi of crystallization; as it progresses, these centers enlarge and fuse with each other. In this fashion, an increasingly large region of the matrix is dehydrated and calcified.

Bone Remodeling

In the adult, bone development is balanced with bone resorption as bone is remodeled to meet stresses placed on it.

In a young person, bone development exceeds bone resorption because new haversian systems are being developed much faster than old ones are being resorbed. Later, in adulthood, when the epiphyseal plates close and bone growth has been attained, new bone development is balanced with bone resorption.

Growing bones largely retain their general architectural shape from the beginning of bone development in the fetus to the end of bone growth in the adult. This is accomplished by surface remodeling, a process involving bone deposition under certain regions of the periosteum with

concomitant bone resorption under other regions of the periosteum. Similarly, bone is being deposited in certain regions of the endosteal surface, whereas it is being resorbed in other regions. The bones of the calvarium are being reshaped in a similar way to accommodate the growing brain; however, how this process is regulated is unclear.

Cortical bone and cancellous bone, however, are not remodeled in the same fashion, probably because osteoblasts and osteoprogenitor cells of cancellous bone are located within the confines of bone marrow and, therefore, they are under the direct, paracrine influence of nearby bone marrow cells. The factors produced by these bone marrow cells include interleukin-1 (IL-1), tumor necrosis factor, colony-stimulating factor-1, osteoprotegrin (OPG), osteoprotegrin ligand (OPGL), and transforming growth factor-β. The osteoprogenitor cells and osteoblasts of compact bone are located in the cellular layer of the periosteum and in the lining of haversian canals and thus are too far from the cells of bone marrow to be under their paracrine influence. Instead, these cells of compact bone respond to systemic factors, such as calcitonin and parathyroid hormone.

The internal structure of adult bone is continually being remodeled as new bone is formed and dead and dying bone is resorbed; for example,

▪

Haversian systems are continually being replaced.

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Bone must be resorbed from one area and added to another to meet changing stresses placed on it (e.g., weight, posture, fractures).

As haversian systems are resorbed, their osteocytes die; in addition, osteoclasts are recruited to the area to resorb the bone matrix, forming absorption cavities. Continual osteoclastic activity increases the diameter and length of these cavities, which are invaded by blood vessels. At this point, bone resorption ceases and osteoblasts deposit new concentric lamellae around the blood vessels, forming new haversian systems. Although primary bone is remodeled in this fashion, which strengthens the bone by ordered collagen alignment about the haversian system, remodeling continues throughout life as resorption is replaced by deposition and the formation of new haversian systems. This process of bone resorption, followed by bone replacement, is known as coupling. The interstitial lamellae observed in adult bone are remnants of remodeled haversian systems.

Bone Repair

Bone repair involves both intramembranous and endochondral bone formation.

A bone fracture causes damage and destruction to the bone matrix, death to cells, tears in the periosteum and endosteum, and possible displacement of the ends of the broken bone (fragments). Blood vessels are severed near the break, and localized hemorrhaging fills in the

zone of the break, resulting in blood clot formation at the site of injury. Soon the blood supply is shut down in a retrograde fashion from the injury site back to regions of anastomosing vessels, which can establish a new circulation route. As a consequence there is a widening zone of injury, on either side of the original break, resulting in a lack of a blood supply to many haversian systems, thus causing the zone of dead and dying osteocytes to increase appreciably. Because bone marrow and the periosteum are highly vascularized, the initial injury site in either of these two areas does not grow significantly, nor is there a notable increase in dead and dying cells much beyond the original injury site. Whenever the bone's haversian systems are without a blood supply, osteocytes become pyknotic and undergo lysis, leaving empty lacunae.

The blood clot filling the site of the fracture is invaded by small capillaries and fibroblasts from the surrounding connective tissue, forming granulation tissue. A similar event occurs in the marrow cavities as a clot forms; the clot is soon invaded by osteoprogenitor cells of the endosteum and multipotential cells of the bone marrow, forming an internal callus of bony trabeculae within a week or so ( Fig. 7-20 ). Within 48 hours after injury, osteoprogenitor cells build up because of increased mitotic activity of the osteogenic layer of the periosteum and the endosteum and from undifferentiated cells of the bone marrow. The deepest layer of proliferating osteoprogenitor cells of the periosteum (those closest to the bone), which are in the vicinity of capillaries, differentiate into osteoblasts and begin elaborating a collar of bone, cementing it to the dead bone about the injury site.

Figure 7-20

Events in bone fracture repair.

Although the capillaries are growing, their rate of proliferation is much slower than that of the osteoprogenitor cells; thus the osteoprogenitor cells in the middle of the proliferating mass are now without a profuse capillary bed. This results in lowered oxygen tension, and these cells become chondrogenic cells, giving rise to chondroblasts that form cartilage in the outer parts of the collar.

The outermost layer of the proliferating osteoprogenitor cells (those adjacent to the fibrous layer of the periosteum), having some capillaries in their midst, continue to proliferate as osteoprogenitor cells. Thus, the collar exhibits three zones that blend together: (1) a layer of new bone cemented to the bone of the fragment, (2) an intermediate layer of cartilage, and (3) a proliferating osteogenic surface layer. In the meantime, the collars formed on the ends of each fragment fuse into one collar, known as the external callus, leading to union of the fragments. Continued growth of the external collar is derived mainly from proliferation of osteoprogenitor cells and, to some degree, from interstitial growth of the cartilage in its intermediate zone.

The cartilage matrix adjacent to the new bone formed in the deepest region of the collar becomes calcified and is eventually replaced with cancellous bone. Ultimately, all of the cartilage is replaced with primary bone by endochondral bone formation.

Once the fragments of bone are united by bridging with cancellous bone, it is necessary to remodel the injury site by replacing the primary bone with secondary bone and resolving the callus.

The first bone elaborated against injured bone develops by intramembranous bone formation, and the new trabeculae become firmly cemented to the injured or dead bone. Matrices of dead bone, located in the empty spaces between newly developing bony trabeculae, are resorbed, and the spaces are filled in by new bone. Eventually, all of the dead bone is resorbed and replaced by new bone formed by the osteoblasts that invade the region. These events are concurrent, resulting in repair of the fracture with cancellous bone surrounded by a bony callus.

Through the events of remodeling, the primary bone of intramembranous bone formation is replaced with secondary bone, further reinforcing the mended fracture zone; at the same time, the callus is resorbed. It appears that the healing and remodeling processes at the fracture site are in direct response to the stresses placed on it; eventually, the repaired zone is restored to its original shape and strength. It is interesting that bone repair involves cartilage formation and both intramembranous and endochondral bone formation.

If segments of bone are lost or damaged so severely that they have to be removed, a “bony union” is not possible; that is, the process of bone repair cannot occur because a bony callus does not form. In cases of this sort, a bone graft is required. Since the 1970s, bone banks have become available to supply viable bone for grafting purposes. The bone fragments are harvested and frozen to preserve their osteogenic potential and are then utilized as transplants by orthopedic surgeons. Autografts are the most successful because the transplant recipient is also the donor. Homografts are from different individuals of the same species and may be rejected because of immunological response. Heterografts, grafts from different species, are least successful, although it has been shown that calf bone loses some of its antigenicity after being refrigerated, making it a worthy bone graft when necessary.

CLINICAL CORRELATIONS

Histophysiology of Bone

Bone supports soft tissues of the body and protects the central nervous system and hemopoietic tissue. It also is the site for attachment of the tendons of muscle that use the bone as levers to increase the mechanical advantage needed for locomotion. Just as important, bone serves as a reservoir of calcium and phosphate for maintaining adequate levels of these elements in the blood and other tissues of the body.

Maintenance of Blood Calcium Levels

Calcium is vital for the activity of many enzymes and also functions in membrane permeability, cell adhesion, blood coagulation, nerve impulse transmission, muscle contraction, among other bodily processes. To fulfill all of the necessary functional requirements for which calcium is responsible, a tightly controlled blood plasma concentration of 9 to 11 mg/dL must be maintained.

Because 99% of the calcium in the body is stored in bone as hydroxyapatite crystals, the remaining 1% must be available for mobilization from the bone on short notice. Indeed, there is a constant turnover between the calcium ions in bone and in blood. The calcium ions retrieved from bone to maintain blood calcium levels come from new and young osteons, where mineralization is incomplete. Because bone remodeling is constant, new osteons are always forming where labile calcium ions are available for this purpose. It seems that older osteons are more heavily mineralized; because of this, their calcium ions are less available.

Hormonal Effects

Osteoclastic activity is necessary for maintaining a constant supply of calcium ions for the body. Parenchymal cells of the parathyroid gland are sensitive to the blood calcium level; when calcium levels fall below normal, parathyroid hormone (PTH) is secreted. As discussed earlier, this hormone activates receptors on osteoblasts, suppressing matrix formation and initiating manufacture and secretion of OPGL and osteoclast-stimulating factor by the osteoblasts. These factors induce osteoclast formation and stimulate quiescent osteoclasts to become active, leading to bone resorption and the release of calcium ions.

Parafollicular cells (C cells) of the thyroid gland also monitor calcium ion levels in the plasma. When calcium ion levels become elevated, these cells secrete calcitonin, a polypeptide hormone that activates receptors on osteoclasts, inhibiting them from resorbing bone. Additionally, osteoblasts are stimulated to increase osteoid synthesis and calcium deposition is increased.

The growth hormone somatotropin, secreted by cells in the anterior lobe of the pituitary gland, influences bone development via somatomedins (insulin-like growth factors), especially stimulating growth of the epiphyseal plates. Children deficient in this hormone exhibit dwarfism, whereas persons with an excess of somatotropin in their growing years display pituitary gigantism.

Many additional factors are involved in bone metabolism, only a few of which are indicated in the following list. Moreover, many of these factors are released by a variety of cells and have numerous target cells; however, only their bone-related functions are listed:

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Interleukin-1, released by osteoblasts, activates osteoclast precursors to proliferate; it also has an indirect role in osteoclast stimulation.

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Tumor necrosis factor, released by activated macrophages, acts in a fashion similar to interleukin-1.

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Colony-stimulating factor-1, released by bone marrow stromal cells, induces osteoclast formation.

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OPG inhibits osteoclast differentiation.

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Interleukin-6, released by various bone cells, especially by osteoclasts, stimulates the formation of other osteoclasts.

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Interferon-γ, released by T lymphocytes, inhibits differentiation of osteoclast precursors into osteoclasts.

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Transforming growth factor-β liberated from bone matrix during osteoclasia, induces osteoblasts to manufacture bone matrix and enhances the process of matrix mineralization; also, it inhibits proliferation of osteoclast precursors and their differentiation into mature osteoclasts.

Acromegaly occurs in adults who produce an excess of somatotropin, causing an abnormal increase in bone deposition without normal bone resorption. This condition creates thickening of the bones, especially those of the face, in addition to disfiguring soft tissue.

CLINICAL CORRELATIONS

Skeletal maturation is also influenced by hormones produced in the male and female gonads. Closure of the epiphyseal plates is normally rather stable and constant and is related to sexual maturation. Precocious sexual maturation stunts skeletal development because the epiphyseal plates are stimulated to close too early. In other people whose sexual maturation is retarded, skeletal growth continues beyond normal limits because the epiphyseal plates do not close.

Osteoporosis affects about 10 million chronically immobilized Americans. It often affects women older than 40 years of age, especially postmenopausal women. Osteoporosis is related to decreasing bone mass, which becomes more serious after menopause, when estrogen secretion drops appreciably. Binding of estrogen to specific receptors on osteoblasts activates the cells to manufacture and secrete bone matrix. With diminished secretion of estrogen, osteoclastic activity is greater than bone deposition, potentially reducing bone mass to the point at which the bone cannot withstand stresses and breaks easily. For decades estrogen replacement therapy coupled with calcium supplements and pain-killers were used to alleviate or eliminate this condition. However, in 2004 it was determined that estrogen replacement therapy increases the risk for heart disease, stroke, breast cancer, and blood clots. A newly developed group of drugs called the

bisphosphonates reduces the incidence of osteoporosis fractures without the risks of estrogen replacement therapy. An early diagnostic tool, dual-energy x-ray absorptiometry (DEXA), is being employed as a reliable method for assessing increasing bone density even in individuals with osteoporosis.

CLINICAL CORRELATIONS

Nutritional Effects

Normal bone growth is sensitive and dependent on several nutritional factors. Unless a person's intake of protein, minerals, and vitamins is sufficient, the amino acids essential for collagen synthesis by osteoblasts are lacking and collagen formation is reduced. Insufficient intake of calcium or phosphorus leads to poorly calcified bone, which is subject to fracture. A deficiency of vitamin D prevents calcium absorption from the intestines, causing rickets in children. Vitamins A and C are also necessary for proper skeletal development ( Table 7-4 ).

TABLE 7-4

Vitamins Affecting Skeletal Development

Deficiency/Excess Effect

Vitamin A deficiency

Inhibits proper bone formation as coordination of osteoblast and osteoclast activities fails; failure of resorption and remodeling of cranial vault to accommodate the brain results in serious damage to the central nervous system

Hypervitaminosis A

Erosion of cartilage columns without increases of cells in proliferation zone; epiphyseal plates may become obliterated, ceasing growth prematurely

Vitamin C deficiency

Mesenchymal tissue is affected because connective tissue is unable to produce and maintain extracellular matrix; deficient production of collagen and bone matrix results in retarded growth and delayed healing (scurvy)

Vitamin D deficiency

Ossification of epiphyseal cartilages is disturbed; cells become disordered at metaphysis, leading to poorly calcified bones, which become deformed by weight bearing (in children, termed rickets; in adults, osteomalacia)

Rickets is a disease in infants and children who are deficient in vitamin D. Without vitamin D, the intestinal mucosa cannot absorb calcium even though there may be adequate dietary intake. This results in disturbances in ossification of the epiphyseal cartilages and disorientation of the cells at the metaphysis, giving rise to poorly calcified bone matrix. Children with rickets display deformed bones, particularly in the legs, simply because the bones cannot bear their weight. Osteomalacia, or adult rickets, results from prolonged deficiency of vitamin D. When this occurs, the newly formed bone in the process of remodeling does not calcify properly. This condition may become severe during pregnancy because the fetus requires calcium, which must be supplied by the mother. Scurvy is a condition resulting from a deficiency of vitamin C. One effect is deficient collagen production, causing a reduction in formation of bone matrix and bone development. Healing is also delayed.

CLINICAL CORRELATIONS

Joints

Bones articulate or come into close proximity with one another at joints, which are classified according to the degree of movement available between the bones of the joint. Those that are closely bound together with only a minimum of movement between them are called synarthroses; joints in which the bones are free to articulate over a fairly wide range of motion are classified as diarthroses.

There are three types of synarthrosis joints according to the tissue making up the union:

1

Synostosis. There is little if any movement, and joint-uniting tissue is bone (e.g., skull bones in adults).

2

Synchondrosis. There is little movement, and joint-uniting tissue is hyaline cartilage (e.g., joint of first rib and sternum).

3

Syndesmosis. There is little movement, and bones are joined by dense connective tissue (e.g., pubic symphysis).

Most of the joints of the extremities are diarthroses ( Fig. 7-21 ). The bones making up these joints are covered by persistent hyaline cartilage, or articular cartilage. Usually, ligaments maintain the contact between the bones of the joint, which is sealed by the joint capsule. The capsule is composed of an outer fibrous layer of dense connective tissue, which is continuous with the periosteum of the bones, and an inner cellular synovial layer, which covers all nonarticular surfaces. Some prefer to call this a synovial membrane.

Figure 7-21

Anatomy of a diarthrodial joint.

Two kinds of cells are located in the synovial layer:

1

Type A cells are macrophages displaying a well-developed Golgi apparatus and many lysosomes but only a small amount of RER. These phagocytic cells are responsible for removing debris from the joint space.

2

Type B cells resemble fibroblasts, exhibiting a well-developed RER; these cells are thought to secrete the synovial fluid.

Synovial fluid contains a high concentration of hyaluronic acid and the glycoprotein lubricin combined with filtrate of plasma. In addition to supplying nutrients and oxygen to the chondrocytes of the articular cartilage, this fluid has a high content of hyaluronic acid and lubricin that permits it to function as a lubricant for the joint. Moreover, macrophages in the synovial fluid act to phagocytose debris in the joint space.